Peptides and proteins in living organisms are frequently modified by additional chemical reactions that occur after their initial formation. These post-translational modifications (PTMs) include but are not limited to phosphorylation, acetylation, methylation, citrullination, crotonylation, butyrylation, ubiquitination, and Proline cis-trans isomerization. PTMs modulate the biological behaviors of the modified peptides and proteins and constitute an important family of biological control mechanisms. The enzymes that install and remove PTMs, as well as the modified peptides and proteins themselves, are frequently implicated as the causative agents of a wide variety of human diseases. PTMs of proteins involved in gene regulation and their associated enzymes are especially important in human cancer. Examples include the tumor suppressor protein p53, whose activities are controlled by lysine methylation and ubiquitination, as well as histones. Histones are proteins that bind to all DNA in the nucleus and create a condensed package of nucleic acids and their associated proteins called chromatin.
Histones are modified by various PTMs, most of which participate in signaling pathways that control the expression of the genes encoded in the associated DNA. The study of PTMs of histones in particular has led to the understanding that a core regulator of gene expression is a “histone code” that comprises multiple PTMs to the histones that are associated with a given DNA sequence. Gene (mis)regulation by epigenetic mechanisms is of critical importance in a large number of disease states, and the mechanisms are best understood in the area of cancer. The activities of various enzymes that install PTMs, enzymes that remove PTMs, and of the PTMs themselves are implicated as causative agents of cancer and are considered to be promising targets for new cancer therapies. Drugs that target histone acetylation (e.g. Vorinostat, a histone deacetylase inhibitor used clinically for the treatment of lymphoma) have already demonstrated that epigenetic gene regulation mechanisms are valid targets for cancer therapy.
Antibodies are the cornerstone of almost all efforts to “read the histone code,” i.e. to analyze the post-translational states of epigenetic/histone targets for both diagnostic purposes and for use in enzyme assays. Antibodies can be raised against almost any peptide sequence, including one containing any specific post-translational modification of interest.
For applications broadly related to diagnostics, antibodies are the molecules used for identifying post-translationally modified peptides or proteins from complex mixtures (e.g. by use in western blots). For applications broadly related to enzyme assays, antibodies are the molecules used to identify and quantify products and/or starting materials in reaction mixtures after varying periods of time.
Despite their dominant position as biochemical tools, antibodies against histone PTM targets have several known shortcomings. Problems include high batch-to-batch variability, high costs, inherently poor selectivity between similar analytes (e.g. those bearing trimethyllysine at different positions on a single protein), and a high rate of failed specificity tests that has been documented to be as high as 25%. But the most serious problem that “quality control” can do little about is epitope masking—the mis-identification of analytes when an antibody misses its target residue because of the proximity of a neighboring residue that also bears a PTM. This problem is intrinsic to the antibody-based analysis of peptides and proteins that are densely decorated with PTMs, such as histones.
Varieties of assays for the enzymes that add and remove post-translational modifications exist and are used in drug discovery programs that search for therapeutic agents that modulate the enzymes' activities. Two broad varieties of assays are available: discontinuous assays that use a post-reaction treatment to identify and quantify the products of enzyme action; and continuous assays that rely on detection of by-products to report indirectly on the progress of the reaction.
One primary disadvantage of discontinuous assays is that they only provide information on the extent of reaction after a fixed amount of time has passed. They do not allow continuous observation of enzyme rate profiles that are more robust identifiers of inhibitors. Homogeneous variants that rely on antibodies have also been developed, but still suffer from the fact that they are operated in a discontinuous manner.
Continuous assays in this field are many and varied in the biochemical mechanisms by which they report on reaction progress, but typically rely on detection of reaction by-products by coupling into subsequent reactions that produce an optical output. Examples can be chemically coupled to an optical output (such as assays that detect formaldehyde, the by-product of demethylase enzymes, by reaction with a chemical that generates a fluorescent product), or enzymatically coupled to produce an optical output (such as assays that detect S-adenosyl homocysteine, the by-product of all methyltransferases, by inclusion of one or more other enzymes that convert it into a chemical species that produces an optical signal).
There is growing appreciation that the specific methylation states of products and starting materials are highly relevant to drug discovery. For example, one highly oncogenic protein can add methyl groups to H3K27me2, but not H3K27me1 or H3K27me0. The drug target demethylase LSD1 specifically removes methyl groups from p53K370me2 to make p53K370me0 (which inactivates p53 and is oncogenic) but is less active against K370me1 (which has distinct biological function) in vivo. LSD1 also demethylates H3K4me2, but not H3K4me3, to generate oncogenic H3K4me0, but has been reported to act instead on H3K9me2 to produce H3K9me0 in the presence of certain cofactors.
There exists a need in the art for an assay that can report in a continuous, homogeneous manner on both the progression of these enzymes' reactions and the identity of the products. There also exists a need for a method of identifying and characterizing analytes comprising post-translational modifications, such as histones, using antibody-free techniques.